Cal-590 AM,Cell Permeant

matters needing attention

1) Fluorescent dyes all have quenching problems. Please try to avoid light as much as possible to slow down fluorescence quenching

2) Ethoxymethyl (AM) is prone to moisture absorption. After removing the ice, please place it in a dry environment at room temperature before opening it. Due to the trace amount of the reagent, please centrifuge it briefly before opening to ensure that the powder does not fall into the bottom of the solution.

3) Cal-590 , AM will solidify at lower temperatures such as 4 ° C and ice bath, and can be used at the bottom, wall, or cover of the centrifuge tube. It can be incubated at 20-25 ° C for a while until fully melted.

4) Cal-590 , AM is the first time to use. It is recommended to prepare the storage solution for immediate use, divide it into individual doses, and strictly adhere to a ≥-20°c seal for dry explosion and storage to prevent expiration. To ensure good experimental results, try to use it in a short period of time.

5) For your safety and health, please wear laboratory clothes and disposable gloves when operating.
Usage

A. Reagent preparation

1) Preparation of Pluronic F-127 mother liquor: Weigh 100mg of Pluronic F-127 powder, add 500ul of DMSO, and prepare a 20 (w/V) DMS mother liquor. The dissolution process requires heating at 40-50 ° C for 20-30 minutes. Store the solution at room temperature without refrigeration. If there is crystal precipitation, it can be dissolved after reheating without affecting its use.

2) HHBS Buffer (1X Hank's Balanced Salt Solution with 20mM HEPES buffer, pH 7,2-7.4) or other physiological buffer.

B. Operation steps

1) Dissolve Ca1-590 AM in anhydrous DMSO to prepare a storage solution of 2-5mM, or take the prepared Cal-590 AM storage solution out of room temperature for reheating. (For example, if preparing a 4mM mother liquor, 9.8ul anhydrous DMSO needs to be added to 50ug Cal-590 AM)

2) Dilute Cal-590 AM+DMSO storage solution with HHBS or other physiological buffer to 10-20uM staining solution containing 0.04% Pluronic F-127.

[Note 1]: Add a certain amount of 20% Pluronic F-127 solution to the Cal-590 AM+DMSO storage solution, so that the concentration of Purronic F-127 is about 0.04%. Pluronic F-127 can prevent AM probes from aggregating in the solution and promote better probe entry into cells. However, Puronic F-127 can significantly reduce the stability of AM probes, so it is only recommended to add it when preparing the working solution, and it is not recommended to add it to the storage solution for long-term storage

[Note 2]: The recommended concentration for the application of Cal-590 AM in most cells is 4-5uM, and the specific usage concentration needs to be optimized according to experimental requirements. In order to avoid excessive loading causing cytotoxicity, it is recommended to use the lowest probe concentration as much as possible on the basis of obtaining effective results

[Note 3]: Cal-590 AM working solution needs to be prepared and used immediately to avoid repeated freezing

3) [Optional] If there are organic ion transporters present within cells (such as CHO cells), Probenecid (1-2mM) may need to be introduced into the aforementioned staining solution (final concentration in the well is 0.5-1mM) to reduce the leakage level of the de esterification probe

[Note 1]: Our company provides various types of probenecid, including water-soluble probenecid, which can reduce the use of organic solvents.

4) Add an equal volume of staining solution to the cell culture well and incubate at 37 ° C for 60-90 minutes. Then, incubate at room temperature for another 30 minutes

5) Suck off the staining solution and wash the cells 1-2 times with HHBS or other physiological buffer (if necessary, use buffer containing transporter inhibitors such as 1mM probenecid) to remove residual probes.

6) Afterwards, in HHBS or other physiological buffer (if necessary, use a buffer containing a transporter inhibitor such as 1mM probenecid), fluorescence is detected using an appropriate excitation/emission wavelength (see product characteristics: Cal-590 AM excitation and emission spectra).

[Note 1]: For fluorescence microscopy detection, it is recommended to use TRITC/Cy3 filters for detection, and it is recommended to use a black framed/transparent cell culture plate.

[Note 2]: For fluorescence enzyme-linked immunosorbent assay (ELISA) detection, it is recommended to use excitation wavelength of 540nm, emission wavelength of 590nm, and cut-off wavelength of 570nm for detection. It is recommended to use a 96 well plate with a black/transparent background: